slc26a12—A novel member of the slc26 family, is located in tandem with slc26a2 in coelacanths, amphibians, reptiles, and birds

Abstract Solute carrier family 26 (Slc26) is a family of anion exchangers with 11 members in mammals (named Slc26a1‐a11). Here, we identified a novel member of the slc26 family, slc26a12, located in tandem with slc26a2 in the genomes of several vertebrate lineages. BLAST and synteny analyses of various jawed vertebrate genome databases revealed that slc26a12 is present in coelacanths, amphibians, reptiles, and birds but not in cartilaginous fishes, lungfish, mammals, or ray‐finned fishes. In some avian and reptilian lineages such as owls, penguins, egrets, and ducks, and most turtles examined, slc26a12 was lost or pseudogenized. Phylogenetic analysis showed that Slc26a12 formed an independent branch with the other Slc26 members and Slc26a12, Slc26a1 and Slc26a2 formed a single branch, suggesting that these three members formed a subfamily in Slc26. In jawless fish, hagfish have two genes homologous to slc26a2 and slc26a12, whereas lamprey has a single gene homologous to slc26a2. African clawed frogs express slc26a12 in larval gills, skin, and fins. These results show that slc26a12 was present at least before the separation of lobe‐finned fish and tetrapods; the name slc26a12 is appropriate because the gene duplication occurred in the distant past.

the ancestral species but were lost in lineages including mammals and (ii) new genes that were specifically gained in nonmammalian lineages.For example, the human genome contains 13 genes that encode aquaporin (Aqp) water channels aqp0-aqp12.Vertebrate Aqps are distributed in 17 subfamilies (Aqp0-16) (Finn et al., 2014); therefore, humans do not have four aqp genes, aqp13-aqp16 (Chauvigne et al., 2019;Finn et al., 2014) (Note that, in this article, protein name abbreviations of all species are shown with the first letter capitalized, and gene names of all species are shown as lowercase and italicized).Molecular phylogenetic analysis of the solute carrier family 1 (Slc1) of amino acid transporters identified new members, Slc1a8 and Slc1a9, that are not present in mammalian lineages (Gesemann et al., 2010).Large-scale analysis of the glucose transporter family Slc2 revealed Slc2a15-a20, where Slc2a15 is widely distributed in birds, reptiles, amphibians, and teleosts, and Slc2a18-a20 is present in teleosts (Xiong and Lei, 2021).Slc8a4, which encodes Na + -Ca 2+ exchanger 4 (Ncx4), is found in teleosts, amphibians, and reptiles (Spencer et al., 2020).Nine members of the Slc12 family, Slc12a1-a9, are known in mammals (Arroyo et al., 2013, Xiong andLei, 2021).slc12a10, which encodes the Na + -Cl − cotransporter 2 (Ncc2), was originally found in fish (Cutler and Cramb, 2008, Hiroi et al., 2008, Wang et al., 2009) and has also been found in amphibians, non-avian reptiles, and some mammals such as horses (Motoshima et al., 2023).A pseudogene for slc12a10 at the same chromosomal locus has been identified in various mammalian genome databases.The human urea transporter family consists of Slc14a1 (Ut-b) and Slc14a2 (Ut-a), and Ut-c have been found in teleosts, and Ut-3, similar to Ut-c, in cartilaginous fish (Kakumura et al., 2009, Mistry et al., 2005).
Slc26 is a family of anion exchangers, and 11 members, Slc26a1-11, are present in mammals.Slc26 proteins share 12 transmembrane regions and a sulfate transporter anti-sigma factor antagonist (STAS) domain in the intracellular carboxy-terminal region (Alper andSharma, 2013, Mount andRomero, 2004).These transport anions such as Cl − , bicarbonate, sulfate, formate, and oxalate ions.The Slc26a1 protein was first identified as a Na + -independent sulfate transporter and is expressed on the plasma membrane of the liver and renal tubules (Bissig et al., 1994).Whole-exome sequencing of a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in the human Slc26a1 gene (slc26a1), and Slc26a1 activity is a major determinant of sulfate homeostasis in humans (Pfau et al., 2023).Slc26a2 was initially isolated by positional cloning of diastrophic dysplasia and is also called diastrophic dysplasia sulfate transporter (Dtdst) (Hastbacka et al., 1994).Numerous slc26a2 mutations have been identified in human recessive chondrodysplasia syndromes (Alper andSharma, 2013, Jackson et al., 2012).Analysis of mice expressing Slc26a2 mutants has revealed skeletal abnormalities, decreased chondrocyte proliferative activity, and decreased sulfate absorption into chondrocytes (Alper andSharma, 2013, Forlino et al., 2005).Slc26a3 was found to be downregulated in adenoma (Dra), and is localized on the apical membrane of intestinal epithelial cells (Schweinfest et al., 1993).It is also responsible for electroneutral Cl − absorption and HCO 3 − secretion in the intestine (Schweinfest et al., 2006).Slc26a4, also called pendrin, is mutated in approximately half of the patients with Pendred syndrome (Coyle et al., 1996, Sheffield et al., 1996).Slc26a5, also known as prestin, is expressed in the outer hair cells of the cochlea and contributes to the auditory function (Zheng et al., 2000).Slc26a6, also known by the name putative anion transporter-1 (Pat-1) or chloride/formate exchanger (Cfex), contributes to anion metabolism in the intestine and kidney, intestinal oxalate excretion, and regulation of cystic fibrosis transmembrane conductance regulator (Cftr) and pancreatic duct HCO 3 − secretion (Wang et al., 2005, Lohi et al., 2000, Knauf et al., 2001).Slc26 has also been reported in fish, insects, and corals and is widely present in the animal kingdom, contributing to its anion metabolism.In fish, Slc26 members contribute to renal sulfate efflux in seawater fish (Kato et al., 2009, Kato and Watanabe, 2016, Katoh et al., 2006, Watanabe and Takei, 2011), Cl − uptake via the gill in freshwater fish (Bayaa et al., 2009, Hwang et al., 2011, Perry et al., 2009), intestinal bicarbonate secretion in seawater fish (Ando et al., 2014, Genz et al., 2011, Kurita et al., 2008, Ruhr et al., 2015), renal sulfate reabsorption in freshwater fish (Nakada et al., 2005), and intestinal oxalate excretion (Whittamore, 2020).In insects such as Anopheles, Slc26 contributes to anion metabolism in the gut and Malpighian tubes (Hirata, Czapar et al., 2012;Hirata, Cabrero et al., 2012).Corals have three types of Slc26: α, β, and γ (Zoccola et al., 2015).In a comparative analysis of the Slc26 family in vertebrates, we initially found an uncharacterized slc26 homolog present in tandem with slc26a2 in the genome databases of chickens, Xenopus, and coelacanths.In this study, we performed molecular phylogenetic and synteny analyses, and showed that the divergence between the new slc26 gene and other known slc26 genes is ancient.We propose the name slc26a12 for this novel member of the Slc26 family.

| MATERIALS AND METHODS
2.1 | Identification of slc26a12 in vertebrate genome databases slc26a12 was initially identified in coelacanth, western clawed frog, green anole, and chicken as an uncharacterized slc26 gene located in tandem with slc26a2 using genome databases (Table 1).The predicted amino acid sequences and accession numbers of Slc26a1/a2/a12 were obtained from these databases and used as queries for BLAST analysis to determine the presence or absence of Slc26a1/a2/a12 in each vertebrate species.BLASTP and TBLASTN analyses were performed on protein or whole-genome shotgun databases of the 59 vertebrate species listed in Table 1 using the NCBI BLAST server (https:// blast.ncbi.nlm.nih.gov) or Ensembl genome browser (https:// www.ensem bl.org) (Martin et al., 2023), and the amino acid sequences and accession numbers of Slc26a1/a2/a12 in each species were collected.
The presence or absence of slc26a12 was determined using synteny analysis.Genome databases of the vertebrate species listed in Table 1 were browsed using the Ensembl genome browser (https:// www.ensem bl.org) (Martin et al., 2023) or NCBI genome data viewer (https:// www.ncbi.nlm.nih.gov/ genome/ gdv/ ) (Rangwala et al., 2021) and the presence of slc26 gene around the slc26a2 loci was manually examined.

| Phylogenetic analysis and multiple amino-acid sequence alignment
The amino acid sequences and accession numbers of the Slc26 family members of various vertebrate species listed in Figure 2 were collected from the GenBank/EMBL/ DDBJ or Ensembl genome browsers.Some of these sequences were aligned with ClustalW software (Chenna et al., 2003) and ESPript (Robert & Gouet, 2014) was used to produce graphical display of the results.
For the phylogenetic analysis, all sequences listed in Figure 2 were aligned using ClustalW software, and the evolutionary history was inferred using the maximum likelihood method and the JTT matrix-based model (Jones et al., 1992).The tree with the highest loglikelihood (−102217.93) is shown.The percentages of trees in which the associated taxa were clustered together are shown below the branches.The initial tree(s) for the heuristic search were obtained automatically by applying the neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model and then selecting the topology with a superior log likelihood value.The tree was drawn to scale with branch lengths measured as the number of substitutions per site.The analysis included a total of 92 amino acid sequences.There were a total of 1240 positions in the final dataset.Evolutionary analyses were performed using MEGA11 software (Tamura et al., 2021).

| Identification of the slc26a12 pseudogene slc26a12p
This study uses the term "pseudogene" in a broad sense, including all cases in which the gene is predicted to not encode a full-length solute carrier protein.When using "pseudogene" in a strict sense to include only cases in which it does not have any predicted function, the nonfunctionality of pseudogenes can be difficult to define (Tutar, 2012).Therefore, in the broad sense here, the term includes the possibility that pseudogenes have functions other than that of solute carrier proteincoding genes.
The pseudogenization of slc26a12 was also analyzed by aligning the nucleotide sequences of regions corresponding to the protein-coding regions of exons and identifying mutations, insertions, and deletions that generate stop codons or frameshifts.The orthologous and slc26a12 in various vertebrate species.

| Semiquantitative reverse transcription polymerase chain reaction (RT-PCR)
Previously prepared total RNA from African clawed frogs (Motoshima et al., 2023, Tran et al., 2006) was used.First-strand complementary DNA was synthesized from 5 μg of total RNA using the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA) with oligo(dT) primers and analyzed by RT-PCR, as described previously (Motoshima et al., 2023, Tran et al., 2006).cDNA was diluted eight-fold with nuclease-free water and used as a template for PCR with gene-specific primers.The African clawed frog genome contains two sets of chromosomes, so there are two copies of slc26a1, slc26a2, and slc26a12, each on the L and S chromosomes.Primers were designed to amplify both copies, and the sequences and accession numbers are shown in The PCR conditions were as follows: initial denaturation at 94°C for 2 min, 28 cycles (African clawed frog slc26a1, slc26a2, slc26a12, and actb) of 94°C for 15 s (denaturation), 54°C for 30 s (annealing), 72°C for 1 min (extension), and a final extension at 72°C for 7 min.After amplification, 3 μL of the PCR mixture was diluted and loaded onto a microchip electrophoresis system for DNA/RNA analysis (MCE-202 MultiNA; Shimadzu, Kyoto, Japan) using a DNA-2500 reagent kit (Shimadzu), according to the manufacturer's instructions.Electrophoresis results were analyzed using MultiNA Viewer software (Shimadzu).

| Identification of slc26a12 as a novel member of the slc26 family
In the genome databases of coelacanths and nonmammalian tetrapod species, such as the western clawed frog, green anole, and chicken, an uncharacterized slc26 gene, which we will name slc26a12 in this study, was found downstream of the slc26a2 locus (Figure 1a).The uncharacterized slc26 gene was not found around slc26a1 of all species examined and slc26a2 of cartilaginous fishes, mammals, and rayfinned fishes (Figure 1a).
To analyze the evolutionary relationship of the new slc26 gene, the predicted amino acid sequences were aligned with those of various Slc26s from tetrapods (human, chicken, green anole, and western clawed frog), lobe-finned fish (coelacanth), ray-finned fish (three-spined stickleback and spotted gar), cartilaginous fish (little skate and elephant shark), and jawless fish (sea lamprey and inshore hagfish), and a phylogenetic tree was constructed (Figure 2).The new Slc26 of the coelacanth, western clawed frog, green anole, and chicken formed a single branch, which was independent of other Slc26 members, supported by a high bootstrap value (100%).Slc26a1 and Slc26a2 of cartilaginous fish, ray-finned fish, coelacanths, and tetrapods also formed independent branches, supported by high bootstrap values (98 and 81%, respectively).Here, we refer to the new Slc26 as Slc26a12.
In the phylogenetic tree, three branches for Slc26a1, Slc26a2, and Slc26a12 in jawed vertebrates and the Slc26a2-like and Slc26a12-like in jawless fishes formed a single branch independent of the other Slc26 members, with a bootstrap value of 100.This result indicated that Slc26a1, Slc26a2, and Slc26a12 form a subfamily of Slc26.
Figure S1 shows multiple alignment of the amino acid sequences of Slc26a12.The amino acid sequences of Slc26a12 are 45%-47% and 49%-52% identical to those of Slc26a1 and Slc26a2, respectively, and this identity is slightly lower than the 52%-54% identity between Slc26a1 and Slc26a2 (Figure 1b-e).In inshore hagfish, the amino acid sequences of Slc26a12-like are 49% identical to those of Slc26a2-like (Figure 1f).
Recent analyses of the hagfish genome indicate that the 2R whole-genome duplication in vertebrates occurred in the ancestral species of the jawed vertebrates (Yu et al., 2024).Synteny analyses of slc26a1 and slc26a2 showed the presence of phosphodiesterase 6B gene (pde6b) and pde6a in the loci of slc26a1 and slc26a2, respectively.In addition, the 5-hydroxytryptamine receptor 4 gene (htr4) was present in the loci of slc26a1 and slc26a2 in gray bichir and spotted gar and that of slc26a2-like in sea lamprey (Figure 1a).These results suggest that slc26a1 and slc26a2 arose through the vertebrate 2R wholegenome duplication.

| slc26a12 in lobe-finned fishes and amphibians
Preliminary analyses suggest that there are species differences in the distribution of slc26a12 in F I G U R E 2 Phylogenetic analysis of Slc26a1, Slc26a2, and Slc26a12.The amino acid sequences of Slc26a1, Slc26a2, Slc26a12, and the other Slc26 members were aligned using ClustalW software.A phylogenetic tree was constructed using the maximum-likelihood method and MEGA software.Numbers indicate bootstrap values, and the scale bar represents the genetic distance of amino acid substitutions per site.For the Slc26a1/a2/a12 subfamily, the amino acid sequences were obtained from 12 vertebrate species: inshore hagfish, sea lamprey, elephant shark, little skate, spotted gar, zebrafish, three-spined stickleback, coelacanth, western clawed frog, green anole, chicken, and human.For the other Slc26 members, the amino acid sequences were obtained mainly from 8 vertebrate species: inshore hagfish, sea lamprey, elephant shark, spotted gar, zebrafish, western clawed frog, chicken, and human.The amino acid sequences of mouse Slc26a7 and Slc26a10 were included because human slc26a10 is pseudogenized.
vertebrates.Therefore, we performed detailed synteny and BLAST analyses to analyze the presence or absence of slc26a12 in different vertebrate lineages.
The results are summarized in Figures 3 and 4 and detailed below.
In lobe-finned fish, BLAST and synteny analyses showed that coelacanth has slc26a12 whereas West African lungfish does not (Figures 3 and 4; Table 1).Both species harbor slc26a1 and slc26a2.

F I G U R E 3
Presence of slc26a12 as well as slc26a2 and slc26a1 in 58 vertebrate species.The accession number of each sequence is summarized in Table 1.Divergence times were retrieved from the TimeTree database (http:// www.timet ree.org/ ) (Kumar et al., 2017).slc26a12p, pseudogene of slc26a12.1R-3R indicates the timing of whole-genome duplications in early vertebrate evolution.
Among the turtles examined, only the common snapping turtle harbors slc26a12 (Figures 3 and 4; Table 1).The green sea turtle has a pseudogene for slc26a12, slc26a12p, at the slc26a12 locus.This study uses the term "pseudogene" in a broad sense, including all cases in which the gene is predicted not to encode a full-length solute carrier protein as described in Materials and Methods.The red-eared slider turtle, Mexican gopher tortoise, and Chinese softshelled turtle lack slc26a12.All examined turtles have one each of slc26a1 and slc26a2.
3.6 | slc26a2-like and slc26a12-like in jawless fishes Phylogenetic analysis showed that the inshore hagfish and sea lamprey have two and one slc26 genes that belong to the Slc26a1/a2/a12 subfamily, respectively (Figure 2).In this study, the inshore hagfish genes are tentatively referred to as slc26a2-like and slc26a12-like, and the sea lamprey gene is referred to as slc26a2-like.Inshore hagfish Slc26a2-like and sea lamprey Slc26a2-like formed a single branch, indicating an orthologous relationship between the two genes.In the phylogenetic tree shown in Figure 2, Slc26a2-like formed a branch with Slc26a2 of jawed vertebrates with a low bootstrap value (55%) and formed a branch with Slc26a1/Slc26a2 of jawed vertebrates with a low bootstrap value (60%).Therefore, the relationship between jawless fish Slc26a2-like and jawed vertebrate Slc26a1/a2/a12 remains unclear.This result does not rule out the possibility that jawless fish Slc26a2-like and jawed vertebrate Slc26a1/a2/a12 are orthologous.
In inshore hagfish, slc26a2-like and slc26a12-like genes are located close to each other on the same chromosome.In the phylogenetic tree, Slc26a12-like formed a branch with Slc26a12 of jawed vertebrates with a low bootstrap value (54%).Therefore, the phylogenetic analysis did not clarify the relationship between jawless fish Slc26a2-like and jawed vertebrate Slc26a1/a2/a12.

African clawed frogs
The distribution of slc26a1, slc26a2, and slc26a12 expression in African clawed frog tissues was analyzed using semiquantitative RT-PCR (Figure 5).The African clawed frog is a tetraploid species, and each of slc26a1, slc26a2, and slc26a12 was present in both of the L and S chromosomes.Therefore, we designed primers in the conserved regions and simultaneously amplified each gene in both chromosomes.The gene expression patterns were as follows: slc26a1 was highly expressed in the intestine, kidney, and larval kidney and slc26a2 in the larval gill The results of semiquantitative RT-PCR are shown as pseudo-gel images of the PCR products using a microchip electrophoresis system.The singlelane presentation is a feature of the electrophoresis system, and the reactions for each primer set were analyzed on the same electrophoresis run.Primers were designed to amplify both copies of slc26a1, slc26a2, and slc26a12 on the L and S chromosomes.Primer sequences and accession numbers are shown in Table 2. Results for slc26a1 and slc26a2 are shown for comparison; actb (β-actin gene) was used as an internal control.and other various tissues; slc26a12 was highly expressed in the larval gill, skin, and fin and at low levels in other tissues.

| Pseudogenization of slc26a12 in turtles and birds
The presence of slc26a12p in turtles and birds was analyzed by dot plot analysis (Figures 6a-e) and by searching for point mutations, insertions, and deletions that produced in-frame stop codons or frameshifts by aligning the nucleic acid sequences of regions orthologous to the exons of slc26a12 (Figures 6f and S2).The genome sequence of the green sea turtle contains a pseudogene that was homologous to the entire region of slc26a12 in the common snapping turtle and had nucleotide substitutions and deletions that generated in-frame stop codons in exon 2 (Figure 6a, f, and S2).
The genome sequence of the burrowing owl contains a pseudogene homologous to the 5′ flanking region, exon 1, intron 1, and 3′ flanking region of slc26a12 in the Bengalese finch (Figure 6b).Deletion of exon 2 in the pseudogene was also observed in other owls, such as the Eurasian eagle and northern spotted owls (Figures 6f and  S2).
The little egret's genome sequence contains a pseudogene homologous to the entire region of slc26a12 in Dalmatian pelicans (Figure 6c).The little egret slc26a12p has a mutation and a partial deletion that generated an in-frame stop codon and frameshift, respectively, in exon 2 (Figure 6f and S2).
The genome sequence of the Emperor penguin contains a pseudogene that is homologous to the 5′ flanking region, exon 1, intron 1, 5′ and 3′ parts of exon 2, and 3′ flanking region of slc26a12 in the Dalmatian pelican (Figure 6d).Deletion of part of exon 2 in the pseudogene was also observed in other owls, such as the rockhopper penguin, yellow-eyed penguin, Adelie penguin, and Magellanic penguin (Figures 6f and S2).In addition, several point mutations that generated in-frame stop codons were observed in both exons 1 and 2 of slc26a12p in penguins, and some of the in-frame stop codons were conserved among slc26a12p in penguins (Figures 6f and  S2).
The mallard genome sequence contains a pseudogene that was homologous to the entire region of slc26a12 in chickens (Figure 6e).Several point mutations that generated stop codons were observed in both exons 1 and 2 of slc26a12p in ducks, such as mallard, duck, Muscovy duck, pink-footed goose, swan goose, and black swan, and some of the in-frame stop codons were conserved among slc26a12p in ducks (Figures 6f and S2).

| DISCUSSION
In this study, we identified a new slc26 gene that is located in tandem with slc26a2 in several vertebrate lineages.Synteny and molecular phylogenetic analyses have confirmed the presence of orthologs of this gene in coelacanths, amphibians, reptiles, and birds (Figure 1).This result indicates that the gene is not a recent duplication in some specific species but was present at least before the divergence of tetrapods and coelacanths.Because this gene appeared in the very distant past, we propose to name it a new member of slc26, slc26a12.Molecular phylogenetic analysis indicated that slc26a12 forms a subfamily along with slc26a1 and slc26a2.The phylogenetic tree also indicated that the distance between Slc26a12 and Slc26a2 within the same species was greater than the distance between Slc26a2s in bony vertebrates and cartilaginous fishes.This result suggests two possibilities.First, gene duplication between slc26a2 and slc26a12 may have occurred before the separation of cartilaginous fish and bony vertebrates.Second, gene duplication between slc26a2 and slc26a12 may have occurred in the common ancestor of coelacanths and tetrapods after the separation of cartilaginous fish and bony vertebrates, and slc26a12 may have evolved at a higher mutation rate than slc26a2.
A comparison of tissue distribution in African clawed frogs revealed that slc26a12 had a different expression pattern than slc26a1, was relatively similar to that of slc26a2, and was most highly expressed in the larval gills, skin, and fin.This result suggests that slc26a12 functions differently from slc26a1 and may have a functional relationship with slc26a2.Its function and physiological role should be clarified in future studies.
slc26a12 is not found in the genome databases of cartilaginous fish, ray-finned fish, or mammals.In mammals, ancestral mammalian species probably lack slc26a12, as other tetrapods possess slc26a12.No pseudogenes for slc26a12 were found in the mammalian genome database.Because some avian and reptilian lineages have lost or pseudogenized slc26a12, the deletion of slc26a12 in mammals is considered to be in line with these lineages.In turtles, many species lack slc26a12, whereas the common snapping turtle has an intact slc26a12 and the green sea turtle has an slc26a12 pseudogene.The Chinese soft-shelled turtle, the oldest divergent turtle species, lacks slc26a12, suggesting that gene deletion and pseudogenization occurred independently in multiple lineages of turtles.In birds, pseudogenes were found in owls, penguins, egrets, and ducks, and these pseudogenes share a common pattern of mutations and deletions in each lineage, but not between the lineages, suggesting that pseudogenes were generated independently in the ancestral species of owls, penguins, egrets, and ducks.Dot plot analyses indicated that slc26a12p in birds shows high homology with intact slc26a12 in related species (Figure 6), suggesting that pseudogenization occurred relatively recently in the bird species.In general, pseudogenes are categorized into three groups: unprocessed duplicated, unprocessed unitary, and processed (Pink et al., 2011;Zhang et al., 2010).Unprocessed and processed pseudogenes are classified depending on whether they contain intron-derived sequences; unprocessed pseudogenes are subcategorized as duplicated and unitary, depending on the presence of a functional parent gene.Dot plot analyses showed that slc26a12p are unprocessed because the homologous sequences were observed in both exon-and intron-coding regions (Figure 6a-e).No functional parent gene was observed for slc26a12p in the avian and reptilian lineages and can thus be categorized as a unitary pseudogene.Most pseudogenes lose the ability to undergo transcription; however, there are examples of pseudogenes that are transcribed (Pink et al., 2011).A duplicated pseudogene can regulate the function of their parent genes via their transcripts (Lin et al., 2007;Scarola et al., 2015).Such mechanisms cannot be postulated in unitary pseudogenes because they lack a parent gene.To determine whether the slc26a12p has any function, further analysis will be required to evaluate in which species and organs it may be transcribed.
There are two possible explanations for why cartilaginous and ray-finned fish lack slc26a12.The first scenario is that slc26a12 is ancient in origin and has been present since before the divergence of the jawless and jawed species.In this case, the ancestors of cartilaginous and rayfinned fish were secondarily deficient in slc26a12.If this scenario is correct, slc26a12-like in hagfish may be an ortholog or closely related gene of slc26a12.The fact that hagfish slc26a12-like is located near slc26a2 on the same chromosome and the results of molecular phylogenetic analysis showed that slc26a12-like and slc26a12 are in a single branch, although the bootstrap value is low, does not exclude this possibility.In this case, lamprey would have lost its secondary slc26a12-like.In a second scenario, slc26a12 may be a paralog acquired by the common ancestors of coelacanths and tetrapods after their divergence from ray-finned fishes.In this case, hagfish slc26a12-like is a uniquely acquired paralog of their lineage.At this point, we find no evidence to confirm or refute either scenario.In both cases, slc26a12 was present before the divergence of coelacanths and tetrapods; thus, it is a relatively ancient gene.
Since Slc26a12 has a primary structure ~50% identical to Slc26a1 and Slc26a2, it may have some function as an anion transporter, which should be clarified in the future study.The physiological significance of the presence of slc26a12 in various lobe-finned fishes and tetrapods and its absence in mammals, ray-finned fishes, and cartilaginous fishes is unknown and remains to be elucidated.All jawed vertebrate species analyzed in this study have one slc26a1 and one slc26a2 without exception, and no species was found to be missing them or to have more than one paralog.This suggests that slc26a1 and slc26a2 are ubiquitously essential for the survival of jawed vertebrates.On the other hand, a difference in the presence or absence of the slc26a12 was observed among the lineages, suggesting that the function of slc26a12 is less important in some lineages, leading to its deletion or pseudogenization.The tissue distribution analysis in African clawed frog indicates that the slc26a2 and slc26a12 are expressed in relatively similar tissues.These results suggest that Slc26a12 may have redundant roles with Slc26a2.It is also possible that Slc26a2 compensates the function of Slc26a12 in lineages that do not have slc26a12.Several teleost groups display secondary loss of the stomach, and some genes involved in gastric acid secretion such as H + /K + -ATPase (atp4a and atp4b), Cl − channel-transporter (slc26a9), and a regulatory subunit of the K + channel (kcne2) have been lost or pseudogenized in agastric (stomachless) fishes (Kato et al., 2024;Castro et al., 2014).Slc26a9 has anion exchange, Cl − channel, and Na + coupled transporter activities and is involved in gastric and pulmonary functions (Chang et al., 2009); the loss or pseudogenization of slc26a9 correlates with the secondary loss of the stomach in several lineages.In the case of slc26a12, the phenotype associated with the loss or pseudogenization of the slc26a12 function is currently unknown.
The ancestors of turtles and birds have slc26a12, which has been pseudogenized in some of these lineages.However, the physiological significance that explains the pseudogenization of slc26a12 in these species is also unclear.Turtles and birds include species with slc26a12p and those with intact slc26a12, both of the species are closely related, and the slc26a12 pseudogenizations are relatively recent events.One possibility is that there is a large functional redundancy between slc26a12 and slc26a2 in these species and the difference between species in the degree of redundancy determines the tolerance for slc26a12 pseudogenization.

NEW & NOTEWORTHY
Slc26 belongs to an anion exchanger family consisting of 11 members in mammals.Based on the analysis of genome databases of various vertebrate species, we report Slc26a12 as a new member of the Slc26 family, which is widely present in coelacanths, amphibians, reptiles, and birds, but not in cartilaginous fishes, ray-finned fishes, or mammals.This study provides an interesting sample for understanding how vertebrates have evolved a family of membrane transporters.
Primers used for polymerase chain reaction amplification of slc26a1, slc26a2, and slc26a12 in the African clawed frog. in vertebrate species.(a) Synteny of slc26a2, slc26a12 and slc26a1 in vertebrates.Conserved synteny of pde6a/pde6b, slc26a1/slc26a2, and htr4 in the genome database of indicated species are also shown.(b-f) Identity in amino-acid sequences of Slc26a1, Slc26a2, and Slc26a12 in chicken (b), green anole (c), western clawed frog (d), and coelacanth (e).(f) Identity in amino-acid sequences of Slc26a2-like and Slc26a12-like in inshore hagfish.

F
Synteny analyses of slc26a12 in various vertebrate species.

F
Pseudogenization of slc26a12 in turtles and birds.Dot plot analyses of slc26a2 and slc26a12 compared to the corresponding genomic regions of green sea turtle (a), burrowing owl (b), little egret (c), emperor penguin (d), and mallard (e).The homologous regions were plotted using Dotmatcher (window size, 20; threshold, 70).(f) Deletion and nonsense mutations of slc26a12p.The schematic representations are based on the dot plot analyses above and a nucleotide sequence alignment in Figure S2.The open reading frames divided into two exons are represented by gray boxes.The deleted regions are indicated by open boxes.Nonsense mutations are indicated by red bars.